This project has as its basic goal an understanding of the structure and the behavior of the mammalian erythrocyte membrane. A major effort has centered attention on those factors contributing to the stability of the plasma membrane and how this may interrelate to the age of the erythrocyte. In addition, work will be continued on the effect of iostonic Tris and isotonic histidine on the intact human erythrocyte. In the former instance, cells incubated in isotonic Tris at 37 degrees swell and show preferential loss of sodium while in the latter instance cells incubated in isotonic histidine, shrink in size and preferentially lose potassium. This effect will be explored further expecially as it relates to the sodium-potassium and calcium ATPase levels, lipid changes and acetylcholine esterase activities. The separation of various age groups of erythrocytes will be explored further using procedures utilizing centrifugation at approximately 15,000 x g at 30 degrees at a high hematocrit. In the main, a study of the top (young) and bottom (old) cells will be undertaken. Preliminary data showed dramatic differences in potassium levels, hemoglobin content, enzymatic activities and susceptibility to Tris swelling in these two groups. Finally, study will be continued on routes to preparation of hemoglobin free membranes (ghosts) reminiscent of the original cell through the use of various reagents such as dimethylethanolamine and choline in the hemolyzing buffers which appear to produce a closed membrane with characteristics similar to the original membrane. It is planned also to continue studies on the importance of a calcium stimulated ATPase to the stability of the membrane.